Fixing agents, method of fixing substance with the same, and substrate having sustance fixed thereto with the same

ABSTRACT

An agent for fixing (a) substance(s), which can fix various substances to be fixed on a substrate via covalent bonds and which has an excellent effect for the prevention of non-specific adsorption is disclosed. A polymer containing a plurality of phosphorylcholine groups and photoreactive groups in one molecule is used. The polymer binds to the substrate and to the substance to be fixed via the photoreactive groups, thereby the substance to be fixed is bound to the substrate via covalent bonds through the polymer, and non-specific adsorption is effectively prevented by the phosphorylcholine groups. Further, an agent for fixing (a) substance(s) for fixing the substance on a substrate, comprising a nonionic water-soluble macromolecule having at least 2 photoreactive groups in one molecule was also provided.

TECHNICAL FIELD

The present invention relates to an agent for fixing (a) desiredsubstance(s) such as polypeptides, nucleic acids lipids and the like ona substrate, a method for fixing (a) substance(s) using the same, and asubstrate on which (a) substance(s) is(are) fixed by the same.

BACKGROUND ART

Immunoplates for immunoassays, on which antibodies or antigens arefixed, DNA chips having chips on which nucleic acids are fixed, and thelike, have been widely used. As one of the conventionally used methodsfor fixing proteins or nucleic acids on substrates, physical adsorptionis widely used. That is, by bringing a hydrophobic substrate such as onemade of polystyrene into contact with an aqueous solution of the proteinor nucleic acid to be fixed, the protein or nucleic acid is fixed on thesubstrate by physical adsorption.

However, the method using physical adsorption has a drawback in that thebinding between the substrate and the fixed substance is weak, so thatthe stability of the substrate on which the substance is fixed isinsufficient. In addition, although blocking of the substrate with aprotein (when fixing a protein) such as bovine serum albumin (BSA),casein, skim milk or the like, or with a DNA (when fixing a DNA) such assalmon sperm DNA or the like is performed in order to preventnon-specific adsorption to the region not covered with the desiredprotein or nucleic acid, the effect for preventing non-specificadsorption by blocking is not necessarily satisfactory.

Fixation of proteins or nucleic acids to substrates is also carried outby covalently binding the functional groups on the substrate with thefunctional groups of the proteins or nucleic acids. However, by thismethod, in cases where the functional group to be used is located in theactive site or in the vicinity thereof of the substance, the activity ofthe substance is lost by the fixation. Further, in cases where anappropriate functional group does not exist, the substance cannot befixed by this method. Still further, although the non-specific bindingis prevented by blocking as in the case of physical adsorption, thepreventive effect is not necessarily satisfactory.

The references disclosing the above-mentioned prior art include thefollowing:

Japanese Laid-open Patent Application (Kokai) No. 11-337551,

Japanese Laid-open Patent Application (Kokai) No. 2001-337089.

On the other hand, it is also known to fix a substance on a substrateusing photoreactive groups (Y. Ito and M. Nogawa, “Preparation of aprotein micro-array using a photo-reactive polymer for a cell adhesionassay,” Biomaterials, 24, 3021-3026 (2003)). In this method, however,attention is not paid to the prevention of non-specific adsorption.

DISCLOSURE OF THE INVENTION

Accordingly, an object of the present invention is to provide an agentfor fixing a substance, which can fix various substances to be fixed ona substrate via covalent bond, and which is excellent in the effect ofprevention of non-specific adsorption, as well as to provide a methodfor fixing a substance using the same and to provide a substrate onwhich a substance is fixed using the same.

The present inventors intensively studied to discover that non-specificadsorption may be effectively prevented by utilizing a water-solublepolymer of which molecule is electrically neutral as a whole as an agentfor binding a substance, thereby completing the present invention.

That is, the present invention provides an agent for fixing (a)substance(s), comprising a water-soluble polymer used for fixing adesired substance on a substrate, which water-soluble polymer has atleast two photoreactive groups in one molecule, the molecule of thewater-soluble polymer being electrically neutral as a whole.

The present invention also provides a method for fixing (a) substance(s)on a substrate, comprising coating the substrate with an aqueoussolution or an aqueous suspension containing the substance to be fixedon the substrate and the agent for fixing (a) substance(s) according tothe present invention; and irradiating the solution or suspension withlight. The present invention further provides a substrate on which saidsubstance was fixed by the method according to the present invention.

By the present invention, an agent for fixing (a) substance(s), whichcan fix various substances to be fixed on a substrate via covalent bond,and which is excellent in the effect of prevention of non-specificadsorption, as well as a method for fixing (a) substance(s) using thesame and a substrate on which a substance is fixed using the same, wasprovided. According to the present invention, (a) substance(s) to befixed may be fixed via covalent bonds irrespective of the type(s) of thesubstance(s), so that a stable fixed substrate may be obtained. Further,by fixing (a) substance(s) with the agent for fixing (a) substance(s)according to the present invention, non-specific binding is effectivelyprevented. Still further, when fixing (a) substance(s) using the agentfor fixing (a) substance(s) according to the present invention,patterning of the fixed substance may be attained by carrying outselective irradiation, so that the substance may easily be fixed to anoptional pattern such as microarray or the like.

BEST MODE FOR CARRYING OUT THE INVENTION

As mentioned above, the agent for fixing (a) substance(s) according tothe present invention comprises a polymer having at least 2photoreactive groups in one molecule, which molecule is electricallyneutral as a whole. The term “molecule is electrically neutral as awhole” herein means that the molecule does not have a group which isionized in aqueous solution having a pH in the vicinity of neutral(pH6-8), or even if the molecule has such groups, it has both of thegroups which become cationic groups and which become anionic groups suchthat the total of the electric charges is substantially 0. The term“substantially” herein means that the total of the electric charges is0, or even if the total is not 0, the total is small enough such thatthe electric charges do not adversely affect the present invention.

The agent for fixing (a) substance(s) according to the present inventionis water-soluble, and the solubility to water (weight in terms of gramwhich can be dissolved in 100 g of water) is preferably not less than 5.

Preferred examples of the water-soluble polymer include the polymerscontaining the unit having the structure represented by the followinggeneral formula [I] and the unit having the structure represented by thefollowing general formula [II].

(wherein in general formulae [I] and [II], X and Y independentlyrepresent a polymerizable atomic group in the polymerized state, R¹represents an atomic group having said photoreactive group, not lessthan 2 units represented by the general formula [I] and not less than 2units represented by the general formula [II] are contained, and thenumber of the units represented by the general formula [I] is largerthan the number of the units represented by the general formula [II].)(The unit having the structure represented by the above-describedgeneral formula [I] may be hereinafter referred to as “unit [I]” forconvenience, and the unit having the structure represented by theabove-described general formula [II] may be hereinafter referred to as“unit [II]” for convenience).

The above-described phosphorylcholine-containing polymer was inventedbased on the conception that non-specific adsorption may be effectivelyprevented by utilizing a macromolecule containing phosphorylcholinewhich is a constituent of biomembrane, paying attention to the fact thatbiomembranes hardly accompany non-specific adsorption in spite of thefact that biomembranes contact various substances.

The unit [I] is a unit containing phosphorylcholine group, and Xrepresents a polymerizable atomic group in the polymerized state. As theX, vinyl monomer residues are preferred. As the unit [I], thoserepresented by the following general formula [V] are preferred:

(wherein X′ represents methacryloxy, methacrylamide, acryloxy,acrylamide, styryloxy or styrylamide group of which vinyl moiety isaddition-polymerized, and R¹ represents single bond or C₁-C₁₀ alkylene(which may be substituted by 1 or 2 hydroxyl group)).

Preferred examples of such a unit include those derived from (i.e.,those monomers in the polymerized state)2-methacryloyloxyethylphosphorylcholine,2-acryloyloxyethylphospholylcholine,N-(2-methacrylamide)ethylphospholylcholine,4-methacryloyloxybutylphospholylcholine,6-methacryloyloxyhexylphospholylcholine,10-methacryloyloxydecylphosphorylcholine,ω-methacryloyldioxyethylenephosphorylcholine and4-styryloxybutylphosphorylcholine. Among these, the unit derived from2-methacryloyloxyethylphosphorylcholine is most preferred.

On the other hand, a preferred example of the photoreactive group in theunit [II] is azide (—N₃) group, but the photoreactive group is notrestricted thereto. As the Y in the unit of the general formula [II],vinyl monomer residues are preferred. Preferred examples of the unit[II] include those represented by the following general formula [VI]:

(wherein Y′ represents methacryloxy, methacrylamide, acryloxy,acrylamide, styryloxy or styrylamide of which vinyl moiety isaddition-polymerized; and R² represents single bond, C₁-C₁₀ alkylene(which may be substituted with 1 or 2 hydroxyl groups) or phenylene(which may be substituted with 1 to 3 substituents selected from C₁-C₄alkyl and hydroxyl groups).

The number of unit [I] is larger than the number of unit [II]. Althoughthe ratio is not restricted, the ratio is preferably about 100:2 to100:50, more preferably about 100:5 to 100:20. Thus, since the polymeris mainly composed of the unit [I] having phosphorylcholine group, thenon-specific adsorption is effectively prevented. The molecular weightof the water-soluble polymer is not restricted, and is usually about1000 to 1,000,000, preferably about 5000 to 100,000.

Although the water-soluble polymer preferably consists of the unit [I]and unit [II] alone, it may contain units derived from otherpolymerizable monomers to the extent that the effect of the presentinvention is not adversely affected. Although the ratio of such otherunits is not restricted as long as the effect of the present inventionis not adversely affected, the ratio is usually not more than 30 mol %,preferably not more than 10 mol % based on the total units in thepolymer.

Preferred examples of the water-soluble polymer include thoserepresented by the following general formula [III]:

(wherein X′, Y′, R¹ and R² represent the same meanings as describedabove, respectively, n and m independently represent integers of notless than 2, and n is larger than m; the units containing X′ and theunits containing Y′ are bound in a random order).

Among the compounds represented by the above-described general formula[III], the polymer represented by the following formula [IV] isespecially preferred:

(wherein n′ represents an integer of 50 to 200, m′ represents an integerof 5 to 40, and the phosphorylcholine-containing units and theazidephenyl group-containing units are bound in a random order).

The above-described water-soluble polymer may be produced by simplypolymerizing the above-described unit [I] and unit [II]. Alternatively,a polymer consisting of the main chain, which does not have the sidechains (phosphorylcholine-containing groups and phosphoreactivegroup-containing groups) is first synthesized, and the side chains arebound later. Still alternatively, the unit [I] having thephosphorylcholine-containing group and the unit [II] which does not havethe photoreactive group are first polymerized, and the photoreactivegroup-containing groups may be bound later. The Examples later describedemploy this method. The polymerization of the monomers and the bindingof the side chains may be easily carried out by those skilled in the artaccording to common technical knowledge, and examples thereof aredescribed in detail in the Examples below.

Preferred examples of the water-soluble polymer used in the presentinvention also include nonionic water-soluble macromolecule (polymer)having at least 2 photoreactive groups in one molecule. Nonionicwater-soluble macromolecules have the effect for preventing non-specificadsorption comparable to the above-describedphosphorylcholine-containing polymers, and have an advantage that theymay be produced or available more inexpensively.

The term “nonionic” herein means that the molecule does notsubstantially have groups which are ionized in aqueous solution having apH in the vicinity of neutral (pH6-8). The term “substantially” hereinmeans that the molecule does not have such a group at all, or even ifthe molecule contains such a group, the amount thereof is so small (forexample, the number of such groups is not more than 1% of the number ofcarbon atoms) that it does not adversely affect the present invention.

Although the molecular weight of the nonionic water-solublemacromolecule is not restricted, it is usually about 350 to 5,000,000,preferably about 500 to several hundred thousands.

Preferred examples of such a nonionic water-soluble macromoleculeinclude polyalkylene glycols such as polyethylene glycol (PEG) andpolypropylene glycol; nonionic vinyl macromolecules containing as theconstituent the following monomers individually or in combination: vinylalcohol, methylvinyl ether, vinylpyrrolidone, vinyloxazolidone,vinylmethyloxazolidone, 2-vinylpyridine, 4-vinylpyridine,N-vinylsuccinimide, N-vinylformamide, N-vinyl-N-methylformamide,N-vinylacetamide, N-vinyl-N-methylacetamide, 2-hydroxyethylmethacrylate, acrylamide, methacrylamide, N,N-dimethylacrylamide,N-iso-propylacrylamide, diacetoneacrylamide, methylolacrylamide,acryloylmorpholine, acryloylpyrrolidine, acryloylpiperizine, styrene,chloromethylstyrene, bromomethylstyrene, vinyl acetate, methylmethacrylate, butyl acrylate, methylcyano acrylate, ethylcyano acrylate,n-propylcyano acrylate, iso-propylcyano acrylate, n-butylcyano acrylate,iso-butylcyano acrylate, tert-butylcyano acrylate, glycidylmethacrylate, ethylvinyl ether, n-propylvinyl ether, iso-propylvinylether, n-butylvinyl ether, iso-butylvinyl ether and tert-butylvinylether; naturally occurring macromolecules such as gelatin, casein,collagen, gum arabic, xanthan gum, gum traganth, guar gum, pullulan,pectin, sodium alginate, hyaluronic acid, chitosan, chitin derivatives,carageenan, starches (carboxymethyl starch and aldehyde starch), dextrinand cyclodextrin; and naturally occurring macromolecules such aswater-soluble cellulose derivatives including methyl cellulose, viscose,hydroxyethyl cellulose, hydroxyethylmethyl cellulose, carboxymethylcellulose and hydroxypropyl cellulose; but the water-soluble nonionicmacromolecules are not restricted thereto. Among these, especiallypreferred are polyethylene glycol, poly(meth)acrylamide (in the presentDescription and Claims, the term “(meth)acryl” means “methacryl” and“acryl”) and poly(glycidyl (meth)acrylate), and polyethylene glycol ismost preferred.

The above-described nonionic water-soluble macromolecules have at least2 photoreactive groups per one molecule. Although the number ofphotoreactive groups per one molecule of the nonionic water-solublemacromolecule is not restricted as long as it is 2 or more, if thenumber is too large, there is a possibility that non-specific adsorptionmay be increased. Therefore, the number of the photoreactive groups perone molecule is preferably not more than 10%, more preferably, not morethan 5% of the number of carbon atoms (excluding the carbons in the sidechains) constituting the macromolecule. A preferred example of thephotoreactive group is azide (—N₃) but the photoreactive group is notrestricted thereto. Specific examples of the photoreactive group includephenyl azide, acetyl and benzoyl; and phenyl azide is especiallypreferred. Although the photoreactive group such as azide group may bedirectly bound to the nonionic water-soluble macromolecule, it may alsobe bound to the nonionic water-soluble macromolecule through anarbitrary spacer structure, the latter being usually easy to prepare andpreferred. In the latter case, the spacer structure is not restricted atall, and examples of the spacer structure include C₁-C₁₀ alkylene (whichmay be substituted with 1 or 2 hydroxyl group) and phenylene (which maybe substituted with 1 to 3 substituents selected from C₁-C₄ alkyl andhydroxyl groups).

Introduction of the photoreactive groups to the nonionic water-solublemacromolecule may easily be carried out based on a conventional method.For example, azide groups may be bound to a nonionic water-solublemacromolecule by reacting a nonionic water-soluble macromolecule havingfunctional groups with an azide compound having a functional group whichreacts with the functional groups of the nonionic water-solublemacromolecule. In cases where polyethylene glycol which is a preferrednonionic water-soluble macromolecule is used, since polyethylene glycolshaving amino groups or carboxyl groups at the both terminals thereof arecommercially available, azide groups may be bound to the polyethyleneglycol by reacting the functional groups of such a commerciallyavailable functional group-containing polyethylene glycol with an azidegroup-containing compound. A plurality of such methods are described inExamples below. Alternatively, in cases where the nonionic water-solublemacromolecule is one formed by polymerization of monomers, such aswater-soluble vinyl macromolecules, a nonionic water-solublemacromolecule having photoreactive functional groups may be produced bycopolymerizing a vinyl monomer which will constitute the mainconstituting units of the water-soluble vinyl macromolecule and aphotoreactive vinyl monomer. Preferred examples of photoreactivewater-soluble vinyl macromolecules includepoly((meth)acrylamide-photoreactive(meth)acrylamide) copolymers,poly(glycidyl(meth)acrylate-photoreactive(meth)acrylamide) copolymers,poly(ethylene glycol mono(meth)acrylate-photoreactive acrylamide)copolymers and the like, and the method for producing these copolymersare described in detail in the Examples below.

The substance to be fixed by using the substance-fixing agent accordingto the present invention is not restricted, and examples thereof includepolypeptides (including glycoproteins and lipoproteins), nucleic acids,lipids, cells (animal cells, plant cells, microorganism cells) andcomponents of the cells (including nuclei, organelles such asmitochondria, membranes such as cell membranes and unit membranes andthe like). When azide group which is used as the photoreactive group inthe substance-fixing agent according to the present invention isirradiated with light, a nitrogen molecule is eliminated andsimultaneously a nitrogen radical is generated. Since this nitrogenradical can bind with not only a functional group such as amino group orcarboxyl group, but also with a carbon atom constituting an organiccompound, the agent according to the present invention can fix most ofthe organic matters.

As the substrate, any substrate may be used as long as at least itssurface is composed of a substance that can bind with theabove-mentioned photoreactive group, and examples of the substrateinclude those made of polystyrene widely used in microplates and thelike, polyethylene terephthalate, polycarbonate, polypropylene and thelike. Glass plates coated with a silane coupling agent may also be used.The shape of the substrate is not restricted at all, and those in theform of plate such as substrates for microarrays, as well as those inthe form of beads, fibers and the like, may be employed. Further, wellsand grooves formed in a plate, such as the wells of microplates may alsobe used. Among these, the substance-fixing agent of the presentinvention is especially suited for microarrays.

Fixation of a desired substance on a substrate using thesubstance-fixing agent according to the present invention may be carriedout as follows. First, an aqueous solution or an aqueous suspensioncontaining the substance to be fixed on the substrate and the agent forfixing (a) substance(s) according to the present invention is applied onthe substrate. In this case, although the concentration (by weight) ofthe substance-fixing agent in the aqueous solution is not restricted,usually it is about 0.005% to 10%, preferably about 0.04% to 1%. Theconcentration (by weight) of the substance to be fixed is usually about10 to 200 times, preferably about 20 to 100 times of that of thesubstance-fixing agent used.

Thereafter, preferably after drying the applied liquid, light isradiated. The light is one which can make the photoreactive group usedyield a radical, and in cases where azide group is employed as thephotoreactive group, UV light is preferred. The dose of the light to beradiated is not restricted, and usually about 1 mW to 100 mW per 1 cm².

By irradiation with the light, the photoreactive groups in the polymeryield radicals, so that the polymer is bound with both the substrate andthe substance to be fixed via covalent bonds. As a result, the substanceto be fixed is fixed on the substrate through the polymer. Since theazide group used as the photoreactive group, upon being irradiated,eliminates a nitrogen molecule and simultaneously yields a nitrogenradical, and this nitrogen radical can bind with not only a functionalgroup such as amino group or carboxyl group, but also with a carbon atomconstituting an organic compound, the agent according to the presentinvention can fix most of the organic matters. By the method accordingto the present invention, since the binding reaction is carried outusing the radicals generated from the photoreactive groups, thesubstance to be fixed is bound not at a particular site thereof, but atrandom sites. Therefore, although some molecules will naturally losetheir activities because their active sites are used for the binding,there are molecules bound at sites which do not affect the active sites.Therefore, by the method of the present invention, even a substancewhich hitherto was difficult to be fixed via covalent bond because anappropriate functional group is located in the active site or a vicinitythereof may be fixed on the substrate via covalent bond without losingthe activity thereof as a whole.

In the regions which were not irradiated with light, since thephotoreactive groups binds with neither the substrate nor the substance,the polymer and the substance are removed by washing. Therefore, bycarrying out selective exposure through a photo mask or the like, thesubstance may be fixed in an arbitrary pattern. Thus, the substance maybe fixed in arbitrary various shapes such as microarray, which is veryadvantageous.

Alternatively, a mixture of the substance-fixing agent according to thepresent invention and the substance to be fixed is microspotted, and theentire surface of the substrate may be irradiated with light.Micro-spotting is a technique which applies liquid on very narrowregions on a substrate, and is widely used in the preparation of DNAchips and the like. Since apparatuses for conducting microspotting iscommercially available, it may easily be carried out by using acommercially available apparatus. Alternatively, a method may beemployed, in which the entire surface of the substrate is coated withthe substance-fixing agent of the present invention, the substance to befixed is microspotted thereon, and the entire of the substrate isirradiated with light. In this case, since spots of the substance to befixed are formed on the layer of the substance-fixing agent, the ratioof the substance fixed on the substrate via covalent bond through thesubstance-fixing agent is increased. Still alternatively, a method maybe employed, in which the substance-fixing agent of the presentinvention is microspotted on the substrate, the substance to be fixed isthen microspotted on the microspots, and then the entire surface of thesubstrate is irradiated. In this case too, since spots of the substanceto be fixed are formed on the layer (in the form of discrete spots) ofthe substance-fixing agent, the ratio of the substance fixed on thesubstrate via covalent bond through the substance-fixing agent isincreased.

The method of the present invention may preferably be employed for thepreparation of immunoassay plates on which antibodies or theantigen-binding fragments thereof, or antigens are fixed, nucleic acidchips and microarrays on which DNAs or RNAs are fixed, and the like, theapplications of the method of the present invention are not restrictedthereto. For example, it may be applied to the fixation of whole cellsor components thereof.

The present invention will now be described more concretely by way ofExamples. The present invention is not restricted to the Examples below.

EXAMPLE 1 Production of Substance-Fixing Agent (Part 1)

According to the following synthesis scheme, a photoreactivemethacryloyloxyethylphosphorylcholine (MPC) polymer was synthesized.

To 2 ml of aqueous PMAc solution (5 wt %=50 mg/mL) (PMAc is the randomcopolymer of 2-methacryloyloxyethylphosphorylcholine (90%) andmethacrylic acid (10%) and is the Compound 1 in the above-describedreaction equation), 12.44 mg of 4-azideaniline and 17.47 mg ofwater-soluble carbodiimide (WSC) were added and then the total volume ofthe mixture was increased to 100 mL with pure water. The mixture wasstirred at pH7 for 24 hours (at 4° C. in a refrigerator). Aftercompletion of the reaction, the resulting mixture was subjected todialysis using a dialysis cassette (produced by PIERCE) until the UVabsorption of azideaniline in the external solution was no longerobserved. Finally, the mixture was lyophilized to obtain photoreactiveMPC polymer.

Test Example 1 Non-Specific Adsorption by Photoreactive MPC Polymer

Non-specific adsorption by the photoreactive MPC polymer prepared inExample 1 was tested as follows by bringing it into contact withFITC-conjugated protein or cells.

(1) Binding of MPC Polymer on Substrate

The photoreactive MPC polymer prepared in Example 1 was dissolved inpure water to a concentration of 10 mg/ml, and 25 μl aliquot thereof wasapplied on a polystyrene substrate (diameter: 2 cm), followed by dryingthe solution in the air. Then UV (wavelength: 260 nm) was selectivelyradiated (irradiance: 16 mW/cm²) thereto through a photo mask having astripe pattern with a width of 100 μm, and the substrate was then washedwith water, thereby fixing the photoreactive MPC polymer on thepolystyrene substrate.

(2) Adsorption of FITC (Fluorescein Isothiocyanate)-Conjugated Protein

On the substrate on which the above-described pattern was formed, 50 μlof an aqueous solution (concentration: 10 mg/ml) of FITC-BSA(commercially available from SIGMA), an aqueous solution (concentration:2 mg/ml) of FITC-IgG (commercially available from SIGMA), or an aqueousFITC-fibrinogen solution (concentration: 3.7 mg/370 μl) was applied andthe resultant was allowed to react at 37° C. for 10 minutes. Afterwashing the resulting substrate with PBS, the substrate was dried andobserved with a fluorescence microscope.

As a result, fluorescence was observed only in the regions on which thephotoreactive MPC polymer was not fixed, and very clear stripe patternwas observed. By this, it was confirmed that MPC polymer does not adsorbproteins non-specifically.

The FITC-fibrinogen used in the above-described test was prepared asfollows. That is, to 20 mg of fibrinogen, 0.1 mg of FITC-I was added andthe mixture was dissolved in 7 ml of 50 mM sodium carbonate buffer(pH8.5). The mixture was allowed to react overnight at 4° C. andconcentrated by centrifugation through a filter having an exclusionmolecular weight of 3000. For desalination, the mixture was centrifuged3 times with MilliQ (trademark) water, and then lyophilized to obtain3.7 mg of FITC-fibrinogen.

(3) Adsorption of Cells

The photoreactive MPC polymer prepared in Example 1 was fixed on apolystyrene substrate with the pattern as described above. After washingthe substrate, the substrate was sterilized with 70% ethanol, and washedwith sterilized water. The resulting substrate was incubated as it is,or after placing 250 μl of 1% BSA/PBS solution or 1% fibrinogen/PBSsolution on the substrate, at 37° C. for 10 minutes. After washing thesubstrate with water, it was subjected to a test for adsorption ofcells. The cells used were Cos 7 cells (commercially available from CellBank of RIKEN) and Raw 246 cells (commercially available from Cell Bankof RIKEN). The cells were scattered on the substrate and observed at 1hour and 4 hours later with a microscope.

As a result, cells were scarcely adsorbed on the regions on which thephotoreactive MPC polymer was fixed, so that the cells were adsorbed inthe form of stripe. Particularly, clear stripe pattern was observed onthe substrates on which photoreactive MPC polymer alone was applied andphotoreactive MPC polymer plus fibrinogen were applied, respectively.

EXAMPLE 2

By the method described below, an immunoassay for detecting collagenusing a collagen-fixed substrate prepared by the method of the presentinvention was carried out.

(i) Aqueous 0.1% solution of the photoreactive MPC polymer (prepared inExample 1) was mixed with aqueous 0.5% collagen solution at a ratio of1:10.

(ii) On a 35 mm polystyrene dish, 3 μl of the MPC/collagen solution wasspotted.

(iii) After drying, fixation was performed by irradiation with UV(wavelength: 260 nm, irradiance: 16 mW/cm², 10 seconds).

(iv) The substrate was washed 3 times with PBS.

(v) As a primary antibody, 100 μl of anti-collagen antibody(commercially available from MONOSAN) (5 μg/ml) or anti-human CD3antibody (commercially available from PHARMINGEN) was spotted, and theresultant was incubated at room temperature for 4 hours.

(vi) After washing the resultant 3 times with PBS, 100 μl ofFITC-labeled secondary antibody (anti-rabbit IgG antibody or anti-mouseIgG antibody) (commercially available from AMERSHAM) (5000-folddiluted), and the resultant was incubated at room temperature for 1hour.

(vii) After washing the resultant 3 times with PBS, the substrate wasobserved with a fluorescence microscope.

(viii) As controls, the same procedures described above were repeatedexcept that MPC polymer alone was spotted, or except that the collagensolution alone was spotted on a Hubble slide glass produced by TAKARA.Further, as controls, the same procedures described above were repeatedexcept that an aqueous solution of azidephenyl group-introducedpolyacrylic acid (PAAc) having negative charge only and collagen, or anaqueous solution of azidephenyl group-introduced polyallylamine (PAAm)having positive charge only and collagen, was spotted on the polystyrenesubstrate.

Results and Discussion

After the immunostaining with the anti-collagen antibody, fluorescencewas observed on the MPC polymer/collagen spots with a fluorescencemicroscope and the spots were not stained with the anti-CD3 antibody, sothat it was confirmed that collagen was stained specifically. In thecontrols, substantially no fluorescence was observed on the spots of MPCpolymer alone, and no fluorescence was observed on the spots ofazidephenyl group-introduced polyacrylic acid (PAAc) having negativecharge alone and collagen. On the spots of azidephenyl group-introducedpolyallylamine (PAAM) having the positive charge only, non-specificstaining occurred irrespective of the type of the antibody. From theabove, the property of MPC polymer that non-specific adsorption is smallwas experimentally proved. Although fixation of collagen on the Hubbleslide glass produced by TAKARA was tried as a control, fluorescence wasnot observed on the spots, and it is thought that collagen could not befixed in this case. From the above, it was proved that proteins can befixed via covalent bonds using MPC polymer, the non-specific adsorptionis inhibited, and so the use of MPC polymer is effective. The resultsare summarized in Table 1 below. TABLE 1 Anti-collagen Antibody Anti-CD3Antibody MPC polymer/collagen fluorescence observed no fluorescence MPCpolymer no fluorescence no fluorescence PAAc/collagen no fluorescence nofluorescence PAAm/collagen fluorescence observed fluorescence observed

EXAMPLE 3 Production of Substance-Fixing Agent (Part 2) (1) Synthesis ofN-(4-azidebenzoyloxy)succinimide

In 40 ml of 1,4-dioxane, 600 mg of 4-azidebenzoic acid (commerciallyavailable from TOKYO KASEI) and 420 mg of N-hydroxysuccinimide(commercially available from WAKO PURE CHEMICALS) were dissolved, and760 mg of N,N′-dicyclohexylcarbodiimide (commercially available fromWAKO PURE CHEMICALS) was added, followed by stirring the resultingmixture at room temperature to start the reaction. Twenty four hourslater, by-product was removed by filtration through a filter paper, andthe solvent of the filtrate was evaporated under reduced pressure. Theobtained product was purified by recrystallization twice from1,4-dioxane and diethyl ether, to obtain 675 mg ofN-(4-azidebenzoyloxy)succinimide (yield: 70.6%).

(2) Introduction of Azide Group into Polyethylene Glycol

In 10 ml of dimethylformamide (commercially available from WAKO PURECHEMICALS), 100 mg of poly(ethylene glycol)bis(3-aminopropyl) terminals(commercially available from ALDRICH, average molecular weight: 1500,hereinafter also referred to as “PEG-NH₂” for convenience) and 68.6 mgof the N-(4-azidebenzyloxy)succinimide prepared in (1) were dissolved,and the mixture was stirred at pH6 at 4° C. for 24 hours. Aftercompletion of the reaction, DMF was evaporated under reduced pressurewith an evaporator, and pure water was added to precipitate theunreacted products. The precipitates were sedimented by centrifugationand the supernatant was recovered, followed by lyophilization thereof toobtain photoreactive PEG-NH₂ (Although the NH₂ groups no longer existbecause they were consumed by the binding with the azide compound, theproduct is referred to such for convenience).

EXAMPLE 4 Production of Substance-Fixing Agent (Part 3)

In 10 ml of pure water, 100 mg of poly(ethyleneglycol)bis(carboxymethyl) ether (commercially available from ALDRICH,average molecular weight: 600, hereinafter referred to as “PEG-COOH” forconvenience), 113.3 mg of 4-azideaniline and 127 mg of water-solublecarbodiimide were dissolved, and the solution was stirred at pH6 at 4°C. for 24 hours. After completion of the reaction, the pH was adjustedto 12 with sodium hydroxide to precipitate the unreacted products. Aftersedimenting the precipitates by centrifugation, the supernatant wasrecovered and desalted with chloroform using a separation funnel (thisoperation was repeated until the pH became neutral), followed bylyophilization of the product to obtain photoreactive PEG-COOH (Althoughthe COOH groups no longer exist because they were consumed by thebinding with the azide compound, the product is referred to such forconvenience).

EXAMPLE 5 Production of Substance-Fixing Agent (Part 4) (1) Synthesis ofN-azidephenylacrylamide

In 10 ml of DMF, 341.2 mg of 4-azideaniline (commercially available fromALDRICH) and 169.1 mg of N-acryloxysuccinimide (commercially availablefrom ALDRICH) were dissolved, and the mixture was stirred at 4° C. tostart the reaction. Twenty four hours later, the product was purified byrecrystallization to obtain N-azidephenylacrylamide.

(2) Synthesis of Photoreactive Poly(acrylamide-N-azidephenylacrylamide)Copolymer

In 30 ml of ethanol, 639.7 mg of acrylamide (commercially available fromWAKO PURE CHEMICALS) and 160 mg of N-azidephenylacrylamide weredissolved, and 82.1 mg of 2,2′-azobisisobutyronitrile (commerciallyavailable from WAKO PURE CHEMICALS) as a polymerization initiator wasadded, followed by stirring the mixture at 60° C. to start the reaction.Five hours later, the polymer was purified by reprecipitation in acetoneand the resultant was dried under reduced pressure to obtainphotoreactive poly(acrylamide-N-azidephenylacrylamide) copolymer.

EXAMPLE 6 Production of Substance-Fixing Agent (Part 5) Synthesis ofPhotoreactive Poly(glycidyl methacrylate-N-azidephenylacrylamide)Copolymer

In 30 ml of methanol, 1.28 g of glycidyl methacrylate (commerciallyavailable from ALDRICH) and 160 mg of N-azidephenylacrylamide weredissolved, and 16.42 mg of 2,2′-azobisisobutyronitrile as apolymerization initiator was added, followed by stirring the mixture at60° C. to start the reaction. Five hours later, the polymer was purifiedby reprecipitation in diethyl ether and the product was dried underreduced pressure to obtain photoreactive poly(glycidylmethacrylate-N-azidephenylacrylamide) copolymer. The epoxy groups wereconverted to glycerol by a treatment with sodium hydroxide (pH 12) tomake the product water-soluble.

COMPARATIVE EXAMPLE 1 Production of Photoreactive Polyacrylic Acid

Photoreactive polyacrylic acid was prepared as follows: In 100 ml ofpure water, 720 mg of polyacrylic acid (commercially available from WAKOPURE CHEMICALS, average molecular weight: 1,000,000), 170.6 mg of4-azideaniline and 1.917 g of water-soluble carbodiimide were dissolved,and the resulting solution was stirred at pH7.0 at 4° C. for 24 hours.After completion of the reaction, the resulting mixture was subjected todialysis until the UV absorption of azideaniline in the externalsolution was no longer observed. Finally, the mixture was lyophilized toobtain photoreactive polyacrylic acid polymer.

COMPARATIVE EXAMPLE 2 Production of Photoreactive Polyallylamine

The same procedures as in Comparative Example 1 were repeated exceptthat the polyacrylamine was used in place of polyacrylic acid to producephotoreactive polyacrylamine.

EXAMPLE 7 Immunoassay

(1) Preparation of Collagen-Fixed Substrate and Immunoassay ofAnti-Collagen Antibody Using the Same

By the procedures described below, using the substance-fixing agentsprepared in Examples 3 to 6, Example 1, Comparative Example 1 andComparative Example 2, respectively, collagen-fixed substrates(polystyrene dish) were prepared, and immunoassays of anti-collagenantibody were carried out using these substrates.

(i) The photoreactive poly(acrylamide-N-azidephenylacrylamide) copolymer(Example 5) or photoreactive poly(glycidylmethacrylate-N-azidephenylacrylamide) copolymer (Example 6) (0.125 wt %)and collagen (0.25 wt %) were mixed at a ratio of 1:1.

(ii) On a 35 mm polystyrene dish, 0.5 μl of the aqueous polymer/collagensolution was spotted.

(iii) After drying, fixation was performed by UV irradiation(wavelength: 260 nm, irradiance: 40 mW/cm², 10 seconds).

(iv) The substrate was washed 3 times with PBS (0.1% Tween 20).

(v) As a primary antibody, 100 μl of 10-fold diluted solution ofanti-collagen antibody (commercially available from MONOSAN) solution(1% BSA/PBS) or 40-fold diluted solution of mouse IgG antibody(commercially available from SANTA CRUZ BIOTECHNOLOGY) was applied inthe form of spots, and the resultant was incubated at room temperaturefor 3 hours.

(vi) After washing the resultant 3 times with PBS (0.1% Tween 20™), 100μl of 20-fold diluted solution of FITC-labeled secondary antibody(anti-rabbit IgG antibody or mouse IgG antibody) (commercially availablefrom AMERSHAM) was added, and the resultant was incubated at roomtemperature for 1 hour.

(vii) After washing the resultant 3 times with PBS (0.1% Tween 20™), thesubstrate was observed with a fluorescence microscope.

(viii) The same procedures were repeated except that a substrate onwhich the photoreactive MPC polymer (Example 1), the photoreactivepolyacrylic acid for comparison (Comparative Example 1), photoreactivepolyallylamine (Comparative Example 2) or the substrate on which thesubstance-fixing agent alone was spotted prepared in each Example wasused.

(2) Results

As shown in Table 2 below, after immunostaining with anti-collagenantibody, fluorescence was observed on PEG-NH₂ (Example 3)/collagenspots, PEG-COOH (Example 4)/collagen spots,poly(acrylamide-N-azidephenylacrylamide) copolymer (Example 5)/collagenspots, poly(glycidyl methacrylate-N-azidephenylacrylamide) copolymer(Example 6)/collagen spots, and fluorescence was not observed on theanti-IgG antibody spots. By this, it was confirmed that collagen wasstained specifically. With the PEG-NH₂ (Example 3) and PEG-COOH (Example4), the fluorescence intensity was about the same as that observed onthe MPC polymer/collagen spots (Example 1) stained with theanti-collagen antibody, and the fluorescence intensity observed on thepoly(acrylamide-N-azidephenylacrylamide) copolymer spots (Example 5) andpoly(glycidyl methacrylate-N-azidephenylacrylamide) copolymer spots(Example 6) was about ½ thereof. In contrast, on the photoreactivepolyacrylic acid having negative charge only/collagen spots (ComparativeExample 1), there was no fluorescence, and on the photoreactiveallylamine having positive charge only/collagen spots (ComparativeExample 2), non-specific staining occurred irrespective of the type ofthe antibody. By these results, it was proved that photoreactivepolyethylene glycol can fix proteins to the degree comparable to the MPCpolymer, and can inhibit non-specific adsorption. With thepoly(acrylamide-N-azidephenylacrylamide) copolymer (Example 5) and withthe poly(glycidyl methacrylate-N-azidephenylacrylamide) copolymer(Example 6), fixation can be attained, although the amount of the fixedprotein is smaller than that attained by using polyethylene glycol(Examples 3 and 4), and non-specific adsorption is inhibited. TABLE 2Substance- Fluorescence Intensity fixing Agent Collagen Anti-collagenAntibody Anti-IgG Antibody Example 3 used 932    25.5 not used 28 29Example 4 used 1483    55.5 not used 62 63 Example 5 used 583  33 notused 26 28 Example 6 used 602  29 not used 30 28 Example 1 used 1185  33not used 30 31 Comparative used 156  39 Example 1 not used 44 27Comparative used 82 95 Example 2 not used 79 90

EXAMPLE 8 Production of Substance-Fixing Agent (Part 6) 1. Synthesis ofN-azidephenylacrylamide

In 97 ml of pure water, 300.02 mg of 4-azideaniline hydrochloric acidsalt (commercially available from ALDRICH) was dissolved, and 3 ml ofaqueous 2 mol/l NaOH solution was added thereto. To the solution, 200 mgof acrylic acid chloride ((commercially available from TOKYO KASEI)diluted with dichloromethane to a volume of 13 ml was slowly droppedwhile stirring the mixture, and after the dropping, the mixture wasvigorously agitated after tightly stopping the vessel. The precipitatedsolids were recovered by filtration, and the dichloromethane phase wasseparated after adding 80 ml of chloroform. The separated organic layerwas dried and the solids were recovered. As a result,N-azidephenylacrylamide was obtained.

2. Synthesis of Photoreactive Poly(ethylene glycolmonomethacrylate-N-azidephenylacrylamide) Copolymer

In a 300 ml eggplant type flask, 49.90 mg of N-azidephenylacrylamide and2225.2 mg of PEG (polymerization degree n=8) were dissolved in 43 ml ofethyl acetate, and 6.31 mg of azobisisobutyronitrile (AIBN) was addedthereto. The flask was filled with nitrogen and was tightly stopped,followed by stirring in water bath at 60° C. for 6 hours. After stoppingthe heating and after evaporation of the solvent to some degree, themixture was transferred to diethyl ether, and the resulting mixture wassubjected to sonicator for 30 minutes. The solids were recovered anddried under reduced pressure for 2 hours. As a result, photoreactivepoly(ethylene glycol monomethacrylate-N-azidephenylacrylamide) copolymerwas obtained.

EXAMPLE 9 Preparation of Allergen-Fixed Substrates and Immunoassays ofAnti-Allergen Antibodies Using the Same

Cedar allergen or mite allergen (commercially available from SEIKAGAKUCORPORATION) was dissolved in pure water to a concentration of 0.25 wt%, and the solution was mixed with the photoreactive polymer (0.125 wt%) prepared in Example 1 or Example 8 at a mixing ratio of 1:1 (v/v). Ona polystyrene substrate, 0.5 μl of the mixed allergen/polymer solutionwas spotted. Then the substrate was irradiated with UV (wavelength: 260nm, irradiance: 15 mW/cm²) to fix the allergen on the polystyrenesubstrate.

The substrate was then washed with PBS (0.1% Tween 20), and 100 μl ofanti-allergen antibody (commercially available from SEIKAGAKUCORPORATION) as the primary antibody dissolved in human serum to a levelof 100 μg/ml was placed in the form of spots on the substrate, followedby allowing the resultant to react at room temperature for 2 hours. Thesubstrate was then washed with PBS (0.1% Tween 20), and 10 μl ofHRP-labeled secondary antibody (commercially available from AMERSHAMBIOSCIENCE) 100-fold diluted with 10% BSA/PBS was added on the spots,followed by allowing the resultant to react at room temperature for 1hour. After washing the substrate with PBS (0.1% Tween 20), 10 μl of achemiluminescence reagent ECL ADVANCE (commercially available fromAMERSHAM BIOSCIENCE) was added, and exposed for 30 seconds with a gelphotographing system to obtain an image. The obtained image wasdigitized by an analysis software.

The digitized luminescence intensities are summarized in Table 3 below.

The one which exhibited the highest luminescence intensity at anantibody level of 100 ng/ml was PEG and the copolymer of Example 8. Theone which exhibited low luminescence intensity when the antibody levelwas 0 was also the copolymer of Example 8, so that it was shown that thenon-specific adsorption thereof was small.

By the above-described results, it was shown that the copolymer ofExample 8, by which the fixed amounts of the allergens were large andthe non-specific adsorption was small, is best suited as the matrix usedfor allergen arrays. TABLE 3 Luminescence Intensity Anti-cedar AllergenAnti-Mite Allergen Antibody Level (ng/ml) Antibody Level (ng/ml) 0 100 0100 PEG 4086 22963 1986  19921 Acrylamide 3193 12357 10156  21350Example 1 (MPC) 6829 23348  0  1567 Example 8 1357 19517 798 22096Allergen alone   0  8593  0  213

INDUSTRIAL AVAILABILITY

The present invention relates to an agent for fixing (a) substance(s) bywhich a substance to be fixed on a substrate can be fixed via covalentbond, and which is excellent in the prevention of non-specificadsorption, as well as to a method for fixing (a) substance(s) using thesame and to the substrate on which the substance was fixed using thesame. According to the present invention, (a) substance(s) to be fixedmay be fixed via covalent bonds irrespective of the type(s) of thesubstance(s), so that a stable fixed substrate may be obtained. Further,by fixing (a) substance(s) with the agent for fixing (a) substance(s)according to the present invention, non-specific binding is effectivelyprevented. Still further, when fixing (a) substance(s) using the agentfor fixing (a) substance(s) according to the present invention,patterning of the fixed substance may be attained by carrying outselective irradiation, so that the substance may easily be fixed to anoptional pattern such as microarray or the like. Therefore, the presentinvention is useful for the preparation of various microarrays and thelike.

1. An agent for fixing (a) substance(s), comprising a water-solublepolymer used for fixing a desired substance on a substrate, whichwater-soluble polymer has at least two photoreactive groups in onemolecule, said molecule of said water-soluble polymer being electricallyneutral as a whole.
 2. The agent for fixing (a) substance(s) accordingto claim 1, comprising a unit having the structure represented by thefollowing general formula [I] and a unit having the structurerepresented by the following general formula [II]:

(wherein in general formulae [I] and [II], X and Y independentlyrepresent a polymerizable atomic group in the polymerized state, R¹represents an atomic group having said photoreactive group, not lessthan 2 units represented by the general formula [I] and not less than 2units represented by the general formula [II] are contained, and thenumber of the units represented by the general formula [I] is largerthan the number of the units represented by the general formula [II].)3. The agent for fixing (a) substance(s) according to claim 1 or 2,wherein said photoreactive group is azide group.
 4. The agent for fixing(a) substance(s) according to claim 2 or 3, wherein the ratio of thenumber of said units represented by the general formula [I] to thenumber of said units represented by the general formula [II] is 100:2 to100:50.
 5. The agent for fixing (a) substance(s) according to any one ofclaims 1 to 4, wherein said polymer has a molecular weight of 1000 to1,000,000.
 6. The agent for fixing (a) substance(s) according to any oneof claims 1 to 5, wherein said X and Y are derived from vinyl monomers.7. The agent for fixing (a) substance(s) according to any one of claims1 to 3, which is represented by the following general formula [III]:

(wherein X′ and Y′ independently represent methacryloxy, methacrylamide,acryloxy, acrylamide, styryloxy or styrylamide group, in the state inwhich the vinyl moiety thereof is addition-polymerized; R¹ represents asingle bond or C₁-C₁₀ alkylene (with the proviso that it may besubstituted with 1 or 2 hydroxyl groups), R² represents a single bond orC₁-C₁₀ alkylene (with the proviso that it may be substituted with 1 or 2hydroxyl groups) or phenylene group (with the proviso that it may besubstituted with 1 to 3 substituents selected from C₁-C₄ alkyl andhydroxyl groups); n and m independently represent integers of not lessthan 2, and n is larger than m; the units containing X′ and the unitscontaining Y′ are bound in a random order).
 8. The agent for fixing (a)substance(s) according to claim 7, wherein said unit containing X′ isderived from 2-methacryloyloxyethylphospholylcholine,2-acryloyloxyethylphospholylcholine,N-(2-methacrylamide)ethylphospholylcholine,4-methacryloyloxybutylphospholylcholine,6-methacryloyloxyhexylphospholylcholine,10-methacryloyloxydecylphosphorylcholine,ω-methacryloyldioxyethylenephosphorylcholine or4-styryloxybutylphosphorylcholine.
 9. The agent for fixing (a)substance(s) according to claim 8, wherein said unit represented by thegeneral formula [I] is derived from2-methacryloyloxyethylphosphorylcholine.
 10. The agent for fixing (a)substance(s) according to claim 9, which is represented by the followingformula [IV]:

(wherein n′ represents an integer of 50 to 200, m′ represents an integerof 5 to 40, and the phosphorylcholine-containing units and theazidephenyl group-containing units are bound in a random order).
 11. Theagent for fixing (a) substance(s) according to any one of claims 1 to10, wherein said substance to be fixed on said substrate is selectedfrom the group consisting of polypeptides, nucleic aids, lipids andcells and constituents of the cells.
 12. A method for fixing (a)substance(s) on a substrate, comprising coating said substrate with anaqueous solution or an aqueous suspension containing said substance(s)to be fixed on said substrate and said agent for fixing (a) substance(s)according to any one of claims 1 to 11; and irradiating said solution orsuspension with light.
 13. The method according to claim 12, whereinsaid substance(s) to be fixed on said substrate is(are) selected fromthe group consisting of polypeptides, nucleic aids, lipids, cells andconstituents of the cells.
 14. The method according to claim 12 or 13,comprising selectively radiating said light so as to pattern the regionto which said substance is fixed.
 15. A substrate on which saidsubstance(s) was(were) fixed by the method according to any one ofclaims 12 to
 14. 16. The agent for fixing (a) substance(s) according toclaim 1, wherein said water-soluble polymer is a nonionic water-solublemacromolecule having at least 2 photoreactive groups in one molecule.17. The agent for fixing (a) substance(s) according to claim 16, whereinsaid nonionic water-soluble macromolecule is a polyalkylene glycol,polyvinyl macromolecule or a naturally occurring macromolecule.
 18. Theagent for fixing (a) substance(s) according to claim 17, wherein saidpolyalkylene glycol is polyethylene glycol, said polyvinyl macromoleculeis poly((meth)acrylamide-photoreactive(meth)acrylamide) copolymer orpoly(glycidyl(meth)acrylate-photoreactive(meth)acrylamide) copolymer.19. The agent for fixing (a) substance(s) according to claim 18, whereinsaid nonionic water-soluble macromolecule is polyethylene glycol,poly(acrylamide-photoreactive acrylamide) copolymer or poly(glycidylmethacrylate-photoreactive acrylamide) copolymer.
 20. The agent forfixing (a) substance(s) according to any one of claims 16 to 19, whereinsaid photoreactive group is phenyl azide group.
 21. The agent for fixing(a) substance(s) according to any one of claims 16 to 20, wherein saidnonionic water-soluble macromolecule has a molecular weight of 500 to5,000,000.
 22. The agent for fixing (a) substance(s) according to anyone of claims 16 to 20, wherein said substance(s) to be fixed on saidsubstrate is(are) selected from the group consisting of polypeptides,polysaccharides, nucleic aids, lipids, cells and constituents of thecells.
 23. A method for fixing (a) substance(s) on a substrate,comprising coating said substrate with an aqueous solution or an aqueoussuspension containing said substance(s) to be fixed on said substrateand said agent for fixing (a) substance(s) according to any one ofclaims 16 to 22; and irradiating said solution or suspension with light.24. The method according to claim 23, wherein said substance(s) to befixed on said substrate is(are) selected from the group consisting ofpolypeptides, polysaccharides, nucleic aids, lipids and cells andconstituents of the cells.
 25. A substrate on which said substance(s)was(were) fixed by the method according to any one of claims 16 to 24.